Antagonists to insulin receptor tyrosine kinase inhibitor

ABSTRACT

A method of diagnosing insulin resistance and related disorders is provided. Additionally, methods of treating mammals with insulin resistance and related disorders is provided. The methods employ antagonists to an insulin receptor tyrosine kinase inhibitor protein.

This application is continuation-in-part of International ApplicationNo. PCT/US94/14893, filed Dec. 28, 1994, which is a continuation-in-partof U.S. application Ser. No. 08/182,241 filed Jan. 14, 1994.

FIELD OF THE INVENTION

The present invention relates to agents that neutralize the activity ofinhibitors of insulin receptor tyrosine kinase activity, especiallyPC-1, and their uses in the diagnosis and treatment of diseases anddisorders involving inappropriate insulin receptor tyrosine kinaseinhibitor expression. In particular the invention relates to agentsuseful in detecting or treating diseases or disorders associated with aninappropriate expression of PC-1. In a preferred aspect, the inventionrelates to agents and methods useful in diagnosing the presence ofinsulin resistance in an individual suspected of having insulinresistance or related disorders, in particular, noninsulin dependentdiabetes mellitus. The invention also provides for methods of treatingmammals, preferably humans, who suffer from diseases associated withinappropriate expression of PC-1. In a particular embodiment, theinvention provides for methods of preventing, treating, or suppressingthe effects of inappropriate expression of PC-1, such as insulinresistance and non-insulin dependent diabetes mellitus.

DESCRIPTION OF RELATED ART

Diabetes mellitus in humans is a complex disorder which can besubdivided into two major clinical syndromes. Each category, in turn,subsumes a number of differing etiologies. About 10% of persons withdiabetes have insulin-dependent diabetes mellitus (IDDM). IDDM ischaracterized by selective destruction of insulin-producing β cells,absolute insulin deficiency, youthful onset, and evidence of autoimmunepathogenesis. Noninsulin-dependent diabetes mellitus (NIDDM) is morecommon and often seen in the context of obesity. NIDDM is characterizedby onset in middle age, resistance to the effects of insulin, andrelative insulin deficiency without β cell destruction. The terms Type 1and Type 2 diabetes have been used to refer to IDDM and NIDDMrespectively. It has been suggested, however, that the terms be used asmodifiers of the physiological states, i.e., Type 1 is sometimes used todescribe an immune mediated pathogenic mechanism and Type 2 used todescribe a non-immune mediated pathogenesis. In Type 1 diabetes, forexample, the immune system mediates destruction of beta cells. Usingthis classification there are three major clinal syndromes: Type 1insulin dependent diabetes, 2) Type 1 noninsulin dependent diabetes, and3) Type 2 noninsulin dependent diabetes. The NIDDM stage of Type 1 thenwould describe the late, slower progressing autoimmune onset.

The pathogenesis of Type 2 NIDDM has not previously been clearlyunderstood. Descriptively, three phases can be recognized inindividuals. The first phase is characterized by demonstrable insulinresistance with normal plasma glucose levels and elevated plasma insulinlevels. In the second stage, postprandial hyperglycemia and increasedinsulin resistance characterize the disease. In the third phase, insulinresistance continues and secreted insulin levels decline resulting infasting hyperglycemia. In all stages the plasma levels of insulin do notcorrespond to plasma glucose levels, i.e. relative insulin deficiency isperceived. Type 2 NIDDM is typified, therefore, by insulin resistance,as well as, insulin secretory defects. (Harrison's. Prncpls. of Int.Med., 11th Ed., McGraw-Hill, publisher, New York, N.Y.)

Several recent studies suggest that the presence of insulin resistanceprecedes the onset of NIDDM. Patients with NIDDM secrete insulin, butnot in a normal fashion, and have resistance to both endogenous andexogenous insulin in muscle and other insulin sensitive tissues. Molleret al., N. Eng. J. Med, 325:938 (1991). The presence of insulinresistance suggests that it may be an initial abnormality in thedisease. In the majority of patients with NIDDM, however, the molecularbasis of the insulin resistance is unknown.

Dermal fibroblasts, derived from a patient exhibiting insulin resistanceand NIDDM, produced an inhibitor of insulin receptor tyrosine kinaseactivation. Insulin receptor content was normal in the patient andpurified insulin receptors had normal tyrosine kinase activity. Theinhibitor was shown to be a glycoprotein exhibiting relative specificitytoward insulin receptor tyrosine kinase. Sbraccia et al., Diabetes,40:295 (1991) and Maddux et al., J. Clin. End. Metab., 77:73 (1993).

Insulin Receptor

The cellular response to insulin is mediated through the insulinreceptor, which is a tetrameric protein consisting of two identicalextracellular alpha-subunits which bind insulin and two identicaltransmembrane beta-subunits which have intracellular tyrosine kinaseactivity. Goldfine, Endocr. Rev., 8:235 (1987). When insulin binds tothe alpha-subunit, the beta-subunit tyrosine kinase is activated andinsulin action ensues. Patients with NIDDM have impaired insulinreceptor tyrosine kinase activity in muscle, fibroblast and othertissues. Abnormalities in the sequence of the insulin receptor gene donot appear to be the cause of the decreased kinase activity in the vastmajority of patients examined. Seino et al., Diabetes 39:129 (1990).

Plasma Cell Membrane Glycoprotein PC-1

PC-1 is a class II (cytoplasmic N terminus) membrane glycoprotein; it isthe same protein as liver nucleoside pyrophosphatase/alkalinephosphodiesterase I (NPPase). Rebbe et al., Mol. Immuno., 30:87-93(1993). Monoclonal antibodies recognizing human placental NPPase havebeen prepared (Yano et al., (1987) Biochem. Biophys. Res. Commun.147:1061-1067) and used to purifiy NPPase from human placenta(Funakoshi, I., et al., (1992) 295:180-187). PC-1 has been detected inother cells including: placenta; chondrocytes; epididymis; kidneytubules; salivary ducts; brain capillaries; skin fibroblasts; myelomacells; skeletal muscle; and fat Rebbe et al., Mol. Immuno., 30:87-93)1993). The size of PC-1 is 115-135 kDa, depending on the tissuestudied; PC-1 also exists as a 230-260 kDa dimer. Human PC-1 has beendeduced to have 873 amino acids, and is mapped to the chromosomelocation, 6q22-6q23, Funakoshi et al., Arch. Biochem. Biophys.,295:180-187 (1992). The extracellular domain of PC-1 cleavesphosphosulfate, pyrophosphate, and phosphodiester linkages. PC-1 mayhave threonine-specific protein kinase activity Oda et al., J. Biol.Chem., 266:16791-16795 (1991). PC-1 has been reported to be closelyassociated with the acid fibroblast growth factor receptor Oda et al.,J. Biol. Chem., 266:16791-16795 (1991), and regulated by TGF-β Huang etal., J. Clin. Invest., 94:560-567 (1994).

Current Treatment of Insulin Resistance and Related Disorders

Current treatment regimes for insulin resistance and diabetes includedietary regimes as well as insulin therapy for patients with IDDM andNIDDM patients who do not respond to dietary changes. Dietary regimesare based on calculations of total caloric intake required for idealbody weight as well as decisions based on fractional distribution of thediet between fat, carbohydrate and protein. However, foods of similarweight and protein, carbohydrate, and fat content do not give rise tosimilar postprandial blood glucose levels. Therefore, substitutioncharts often relied on in diet protocols are increasingly questionable.Moreover these dietary charts do not take into account changes inpostprandial blood glucose levels that may result from particularcombinations of ingested foods.

Insulin therapy is required for all patients with IDDM and for patientswith NIDDM that do not respond to dietary changes. There are no standardtreatment protocols for patients with insulin therapy although one ofseveral typical regimes is usually indicated. Conventional insulintherapy involves one or two injections of insulin per day. Therapy isusually conducted on an out patient basis with moderate problems inpatient compliance.

Nontypical treatments exist for diabetes which include pancreatictransplant. In addition, autoimmune regulation of diabetes is beingextensively studied. However the currently available immunosupressivessuch as cyclosporine and FK506 are usually not warranted.

There is a need therefore, for effective agents that can be used in thediagnosis and therapy of individuals with insulin resistance or NIDDMthat offer both ease of use, as well as effective course of therapy.

It is an object of this invention to provide agents useful in variousdiagnostic and therapeutic methods for the detection and treatment ofinsulin resistance and related disorders.

SUMMARY OF THE INVENTION

The present invention relates to the diagnosis and treatment of diseasesand disorders resulting from an inhibition of insulin receptor tyrosinekinase activity. The invention provides for agents useful in detectingor treating diseases or disorders involving inappropriate inhibition ofinsulin receptor tyrosine kinase activity.

The present inventors have discovered that certain individuals withinsulin resistance overexpress an endogenous inhibitor of insulinreceptor tyrosine kinase activity which they have identified as themembrane glycoprotein PC-1. The identification of the inhibitor ofinsulin receptor tyrosine kinase activity has allowed the presentinventors to discover agents and methods useful in detecting andtreating diseases and disorders associated with inhibitors of insulinreceptor tyrosine kinase activity.

Therefore, in one embodiment the present invention provides a method fordetecting or measuring the amount of an insulin receptor tyrosine kinaseinhibitor in a sample comprising the steps of; contacting the samplewith a first anti-inhibitor antibody under conditions which allowimmunospecific binding to occur; (b) contacting the sample with a secondanti-inhibitor antibody under conditions which allow immunospecificbinding to occur; and (c) detecting or measuring any immunospecificbinding that occurs between a component of the sample and both the firstand the second anti-inhibitor antibodies, in which immunospecificbinding of a component of the sample with said first and secondantibodies indicates the presence or amount of the inhibitor in thesample. In a preferred embodiment the insulin receptor tyrosine kinaseinhibitor is PC-1.

In another embodiment, the invention provides for a method for detectingthe overexpression of an insulin receptor tyrosine kinase inhibitor in asample comprising the steps of (a) measuring the total amount of aninsulin receptor tyrosine kinase inhibitor in the sample according tothe methods presented herein and; (b) comparing the amount determined instep (a) to an amount of insulin receptor tyrosine kinase inhibitorpresent in a standard sample, an increased level in the amount of step(a) being indicative of an overexpression of the insulin receptortyrosine kinase inhibitor. In a preferred embodiment the insulinreceptor tyrosine kinase inhibitor is PC-1.

In another preferred embodiment the invention provides for a method fordetecting the presence or the onset of a disease or disorder associatedwith an overexpression of a PC-1 molecule comprising the steps of (a)measuring the amount of PC-1 in a sample according to the methodspresented herein, and (b) comparing the amount of PC-1 in the sample tothe amount of PC-1 in a standard sample, an increase in the amount ofPC-1 in the sample being indicative of a disease or disorder. Theinvention thus provides for a method of diagnosing a disease or disorderassociated with increased levels of an insulin receptor tyrosine kinaseinhibitor, especially PC-1. In preferred aspects, the invention providesfor diagnosing a disease associated with an overexpression of PC-1 suchas insulin resistance, including non-insulin dependent diabetesmellitus.

In further embodiments, the invention provides for kits for assaying forthe detection of increased amounts of an insulin receptor tyrosinekinase inhibitor such as PC-1. The kits of the instant invention areuseful in diagnosing a disease or disorder associated with PC-1expression, such as insulin resistance and non-insulin dependentdiabetes mellitus.

In a further embodiment the invention provides for a method ofneutralizing the effect of an insulin receptor tyrosine kinase inhibitorwhich comprises providing an agent capable of neutralizing the effect ofan inhibitor on insulin receptor tyrosine kinase activity. In oneembodiment the agent is an antibody and the method is performed invitro. In a preferred embodiment the agent is an antibody, preferably amonoclonal antibody, and the method is performed in vivo. Preferably,according to this aspect of the invention, the antibody is compatiblewith the host immune system. In this aspect of the invention, when thesubject is a human the antibody is preferably a human or humanizedantibody.

In another embodiment, the invention provides a method of treating amammal with a disease or disorder associated with PC-1 expression whichcomprises providing for an agent effective in neutralizing the effect ofPC-1 on the insulin receptor tyrosine kinase activity. The agent is, ina preferred embodiment an antibody capable of binding to and preventingthe effect of the PC-1 on insulin receptor tyrosine kinase activity.

In yet another embodiment the invention provides for a pharmaceuticalcomposition comprising an agent capable of neutralizing the effect of aninhibitor of insulin receptor tyrosine kinase activity such as PC-1along with a suitable pharmaceutical excipient.

DESCRIPTION OF THE DRAWINGS

FIG. 1 (SEQ ID NO: 1) shows the amino acid sequence of the insulinreceptor tyrosine kinase inhibitor PC-1.

FIG. 2: FIG. 2 is a comparison of insulin receptor autophosphorylationin fibroblasts from a patient with insulin resistance and NIDDM and anage and sex matched control. The fibroblasts from the patient withinsulin resistance and NIDDM (MW) required significantly more insulin tostimulate autophosphorylation.

FIG. 3: FIG. 3 is polyacrylamide gel showing the inhibitor PC-1 purifiedfrom the fibroblasts of a patient with insulin resistance and NIDDM. Thelane marked WGA is a sample of material purified over a wheat germagglutinin agarose column. The lane marked 1M NaCl elution shows thesame material eluted from an ATP agarose column. The PC-1 elutes as twobands with relative molecular weights of 130 and 260 kDa.

FIGS. 4A-4C:

FIG. 4A is a Western blot analysis of PC-1 content in fibroblasts of apatient with NIDDM and insulin resistance (lane MW), compared withequivalent controls (lanes C1, C2, and C3). The Western blot reveals a5-10 fold increase in the levels of PC-1 in the fibroblasts of thepatient with insulin resistance and NIDDM.

FIG. 4B: FIG. 4B is a Northern blot analysis of PC-1 message infibroblast cells of the patient sample MW and the three control samples(C1, C2, and C3).

FIG. 4C: FIG. 4C is a Northern blot analysis of the same samples as 4Bprobed with cDNA to β-actin showing no change in the level of message.The 8.2 and 3.6 Kb species of the insulin receptor mRNA, and the 2.0 Kbspecies of the actin mRNA are shown.

FIGS. 5A-5C:

FIG. 5A: PC-1 activity in dermal fibroblasts from NIDDM patients andcontrols as determined by hydrolysis of the synthetic substrate3'-phosphoadenosine, 5'-phosphosulfate (PAPS).

FIG. 5B: PC-1 content in dermal fibroblasts from NIDDM patients (D1-D4)and controls (C1-C3) as demonstrated by Western blot analysis. The 130and 260 forms of PC-1 are indicated.

FIG. 5C: Insulin receptor β-subunit autophosphorylation in fibroblastsfrom 2 NIDDM patients (NIDDM 2 and NIDDM 4)and matched controls(control). The results are presented as a bar graph representing theresults obtained from an autoradiograph.

FIGS. 6A-6C.

FIG. 6A: A competition-inhibition plot is shown demonstrating the lackof an effect of PC-1 overexpression in MCF-7 cells on insulin binding.

FIG. 6B: Inhibitory effect of PC-1 overexpression in MCF-7 cells oninsulin stimulated tyrosine kinase activity as demonstrated by Westernblot analysis. MCF-7 cells transfected with PC-1 (MCF-7 PC-1) and MCF-7cells transfected with pRKneo (MCF-7 NEO). The locations of the insulinreceptor β subunit and IRS-1(pp 185)are shown.

FIG. 6C: Effect of PC-1 overexpression in MCF-7 cells on insulinstimulated [³ H]thymidine incorporation. In MCF-7 NEO cells, basalincorporation was 19.9±1.8 (mean±SEM, n=4), and in the presence of 1 mMinsulin was 36.6±4.7. In MCF-7 PC-1 cells basal incorporation was15.0±1.9, and in the presence of 1 mM insulin was 34.2±4.2. Results aremean±SEM of 4 separate experiments.

DEFINITIONS

The term "treatment" in the instant invention is meant to includetherapeutic treatment, as well as prophylactic, or suppressive measuresfor the disease or disorder. Thus, for example, in the case of NIDDM,successful administration of the agent prior to onset of the diseaseresults in "treatment" of the disease. As another example, successfuladministration of the agent after clinical manifestation of the diseaseto combat the symptoms of the disease comprises "treatment" of thedisease. "Treatment" also encompasses administration of the agent afterthe appearance of the disease in order to eradicate the disease.Successful administration of the agent after onset and after clinicalsymptoms have developed, with possible abatement of clinical symptomsand perhaps amelioration of the disease, comprises "treatment" of thedisease.

Those "in need of treatment" include mammals already having the diseaseor disorder, as well as those prone to having the disease or disorder,including those in which the disease or disorder is to be prevented.

A "disease involving inappropriate expression of PC-1" within the scopeof the present invention is meant to include diseases or disorderscharacterized by an overabundance of the membrane glycoprotein PC-1.This overabundance may be due to any cause including, but not limitedto, overexpression at the molecular level, prolonged or accumulatedappearance at the site of action, or increased activity of theglycoprotein relative to normal. Such an overabundance can be measuredrelative to normal expression, appearance, or activity of the PC-1according to, but not limited to, the assays demonstrated herein. Suchdisorders may include insulin resistance, and abnormal glucosetolerance, as well as the many disorders in which insulin resistanceplays a key role such as obesity, diabetes mellitus, ovarianhyperandrogenism, and hypertension. The disease or disorder fortreatment according to the methods presented herein are preferably thosediseases where insulin resistance is present due to an overexpression ofPC-1. Therefore, in a preferred embodiment, the disease or disorder isinsulin resistance, preferably NIDDM, and more preferably Type 2 NIDDM.

The expressions, "agent", "agent that neutralizes the activity of aninsulin receptor tyrosine inhibitor", and "an agent specific for aninhibitor of insulin receptor tyrosine kinase activity" within the scopeof the present invention are meant to include any molecule which blocksor prevents the interaction between PC-1 and the membrane associatedinsulin receptor tyrosine kinase. Such agents accomplish this effect invarious ways. For instance, one class of agents will bind to PC-1 withsufficient affinity and specificity to neutralize PC-1 such that it hasno effect on the insulin receptor tyrosine kinase. Included within thisgroup of agents are antibodies. Another class of agents are moleculesbased on a protein-protein interaction between the inhibitor, such asPC-1, and the insulin receptor. Such molecules include fragments of theinsulin receptor or small bioorganic molecules, e.g. peptidomimetics,that will prevent the interaction between the membrane associatedinsulin receptor and the inhibitor. Non-limiting examples of selectedagents include antibodies, proteins, peptides, glycoproteins,glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleicacids, bioorganic molecules, peptidomimetics, pharmacological agents andtheir metabolites, transcriptional and translation control sequences,and the like. Another class of agents blocks or prevents intracellularor membrane associated events occurring between the insulin receptor andthe insulin receptor tyrosine kinase inhibitor. In a preferredembodiment the agent is an antibody, which antibody has the desirableproperties of binding to an inhibitor such as PC-1 and preventing itsinteraction with the membrane associated insulin receptor. In anotherpreferred aspect the agent is a soluble receptor based on the primarystructure of the insulin receptor which has the desirable qualities ofpreventing the interaction of the membrane associated insulin receptorwith the inhibitor of tyrosine kinase activity while being unable tobind free insulin in vivo. In another preferred embodiment the agent isa bioorganic molecule capable of preventing the interaction between thePC-1 and the insulin receptor. In another aspect of the invention the"agent" is a transcriptional regulator of PC-1 expression.

The term "inhibitor" as used herein is meant to include any moleculewhich, by virtue of its association with the insulin receptor or insulinreceptor signalling pathway brings about an insulin insensitivity. In apreferred aspect of the present invention, the inhibitor is the membraneglycoprotein PC-1 described in Buckley et al., J. Biol. Chem., 265(29):17506-17511 (1990). The corresponding protein has been described inother species, for instance, murine, Van Driel et al., PNAS,82:8619-8623 (1985). The present invention is meant to include the humanmolecules as well as those of other species.

The terms "neutralize", and "neutralize the activity of" are used hereinto mean, for example, block, prevent, reduce, counteract the activityof, or make the inhibitor ineffective by any mechanism. Therefore, theagent may prevent a binding event necessary for inhibition of insulinreceptor tyrosine kinase activity. By "neutralizing antibody" is meantan antibody molecule as herein defined which is able to block orsignificantly reduce an effector function of the inhibitor. For example,a neutralizing antibody may inhibit or reduce the ability of PC-1 tointeract with the insulin receptor to decrease tyrosine kinase activity.Alternatively, the neutralizing antibody may inhibit or reduce theability of PC-1 to block the insulin receptor signalling pathway. Theneutralizing antibody may also immunospecifically bind to the inhibitorsuch as PC-1 in an immunoassay for inhibitor activity such as the onesdescribed herein. It is a characteristic of the "neutralizing antibody"of the invention that it retain its functional activity in both in vitroand in vivo situations. The term "antibody" is used in the broadestsense and specifically covers single anti-inhibitor monoclonalantibodies and anti-inhibitor antibody compositions with polyepitopicspecificity (including neutralizing and non-neutralizing antibodies).The term "antibody" is also meant to include both intact molecules aswell as fragments thereof which bind the inhibitor, such as, forexample, F(ab')₂, Fab', Fab and Fv. These fragments lack the Fc fragmentof an intact antibody molecule, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody,Wahl et al., J. Nucl. Med., 24:316-325 (1983), properties which may bedesirable for particular therapeutic or diagnostic utilities. It will beappreciated that these antigen-binding fragments of the antibodiesuseful in the present invention may be used for the detection andquantitation of inhibitor proteins or peptides as disclosed herein forintact antibody molecules. Such fragments are typically produced byproteolytic cleavage, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab')₂ fragments) or by reducing thedisulfide bridges.

The term "monoclonal antibody" as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen.

The monoclonal antibodies herein include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-inhibitor antibody with a constant domain (e.g."humanized" antibodies), or a light chain with a heavy chain, or a chainfrom one species with a chain from another species, or fusions withheterologous proteins, regardless of species of origin or immunoglobulinclass or subclass designation, as well as antibody fragments [e.g., Fab,F(ab)₂, and Fv], so long as they exhibit the desired biologicalactivity. See, e.g. U.S. Pat. No. 4,816,567 and Mage & Lamoyi, inMonoclonal Antibody Prod. Techniques and Applns, pp.79-97 (MarcelDekker, Inc.), N.Y. (1987).

For example, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler & Milstein, Nature, 256:495 (1975), or may be made by recombinantDNA methods (U.S. Pat. No. 4,816,567). The "monoclonal antibodies" mayalso be isolated from phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990), for example.

"Humanized" forms of non-human (e.g. murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab', F(ab)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from thecomplementary determining regions (CDRs) of the recipient antibody arereplaced by residues from the CDRs of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human FR residues. Furthermore, the humanized antibody may compriseresidues which are found neither in the recipient antibody nor in theimported CDR or FR sequences. These modifications are made to furtherrefine and optimize antibody performance. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR residues are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin.

The term "sample" as used herein, refers to a biological samplecontaining or suspected of containing an insulin receptor tyrosinekinase inhibitor. This sample may come from any source, preferably amammal and more preferably a human. Such samples include aqueous fluidssuch as serum, plasma, lymph fluid, synovial fluid, follicular fluid,seminal fluid, milk, whole blood, urine, cerebrospinal fluid, saliva,sputum, tears, perspiration, mucous, tissue culture medium, tissueextracts, and cellular extracts.

The term "mammal" for the purposes of treatment refers to any animalclassified as a mammal, including but not limited to, humans, sport,zoo, pet and domestic or farm animals such as dogs, cats, cattle, sheep,pigs, horses, and primates, such as monkeys. Preferably the mammal is ahuman.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have identified an inhibitor of insulin receptortyrosine kinase, present in fibroblasts of certain insulin resistantindividuals, as the class II membrane glycoprotein PC-1. The presentinventors have also discovered that PC-1 activity is increased infibroblasts of patients with NIDDM. Based on this discovery, the presentinventors have designed novel methods of diagnosing and treatingdiseases and disorders involving inappropriate expression of membraneglycoprotein PC-1 based on agents which neutralize the activity of PC-1as it effects the insulin receptor. Therefore, the present inventionprovides for agents useful in a number of in vitro and in vivodiagnostic and therapeutic situations. The present invention alsoprovides for methods of using the agents in the diagnosis and treatmentof diseases and disorders associated with inappropriate PC-1 expression.The invention will now be described with respect to these agents. Theinvention will also be illustrated by the methods of using the agents inin vitro and in vivo applications.

1. Agents

In the broadest aspect, the agents of the present invention, by virtueof their interaction with inhibitors of insulin receptor tyrosine kinaseactivity, prevent or block the inhibition of tyrosine kinase activity.The agents of the invention are based on the discovery that PC-1inhibits tyrosine kinase activity in patients with insulin resistance.Therefore, the agents of the invention neutralize the ability of PC-1 toinhibit insulin receptor tyrosine kinase activity. Although the presentinventors do not wish to be bound by scientific theory the agents of theinvention may block or prevent the interaction of the inhibitor with theinsulin receptor tyrosine kinase activity. On the other hand, the agentmay block or prevent the interaction of an inhibitor with the insulinreceptor signalling pathway. Additionally, there are at least twomechanisms by which PC-1 may inhibit insulin action, i.e., dependent oninsulin receptor phosphorylation, or not dependent on insulin receptorphosphorylation. Agents which effect either of these two mechanisms aremeant to be included within the scope of the present invention.

2. Antibody Agents

In a preferred aspect "agent" within the scope of the present inventionis meant to include antibodies. In a preferred embodiment the antibodyof the invention is directed against the inhibitor and, by virtue ofimmunospecific binding prevents the interaction between the inhibitorand the insulin receptor.

3. Preparation of Anti-Inhibitor Antibodies

According to this aspect of the invention antibodies are isolated thatare reactive with PC-1 and additionally block or prevent PC-1interaction with the insulin receptor.

4. Polyclonal antibodies

Polyclonal antibodies to PC-1 molecules or fragments thereof aregenerally raised in animals by multiple subcutaneous (sc) orintraperitoneal (ip) injections of PC-1 or PC-1 fragments and anadjuvant. The full length amino acid sequence of PC-1 is provided inFIG. 1, SEQ ID NO 1. The full length protein or any immuno-dominantfragment can be used as an immunogen. It may be useful to conjugate PC-1or a fragment containing the target amino acid sequence to a proteinthat is immunogenic in the species to be immunized, e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for example,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl₂, or R¹ N═C═NR, where R and R¹are different alkyl groups.

Animals are immunized against the PC-1 polypeptides or fragments,immunogenic conjugates, or derivatives by combining 1 mg or 1 μg of thepeptide or conjugate (for rabbits or mice, respectively) with 3 volumesof Freund's complete adjuvant and injecting the solution intradermallyat multiple sites. One month later the animals are boosted with 1/5 to1/10 the original amount of peptide or conjugate in Freund's completeadjuvant by subcutaneous injection at multiple sites. Seven to 14 dayslater the animals are bled and the serum is assayed for PC-1 or PC-1fragment antibody titer. Animals are boosted until the titer plateaus.Preferably, the animal is boosted with the conjugate of the same PC-1 orPC-1 fragment, but conjugated to a different protein and/or through adifferent cross-linking reagent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

5. Monoclonal antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier"monoclonal" indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the anti-PC-1 monoclonal antibodies of the invention may bemade using the hybridoma method first described by Kohler and Milstein,Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S.Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as herein described to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the PC-1 or PC-1 fragment used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Prncpls. and Practice, pp.59-103, Academic Press, (1986).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Maryland USA.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against PC-1.

6. Screening for Antibody Agents

Screening for antibody agents useful in the context of the presentinvention will usually be a multistep process. In one embodiment, theantibodies of the present invention have the desirable characteristicsof binding the PC-1 molecule with reasonably high affinity andspecificity. Additionally, the antibodies of the invention will,preferably, prevent or block the interaction between PC-1 and themembrane associated insulin receptor such that tyrosine kinase activityof that receptor is not affected. Preferably, screening for usefulantibodies is performed with the goal of isolating antibodies with oneor both the desirable qualities of an antibody agent.

Specific in vitro binding assays, such as the ones described below, canbe used to isolate antibodies reactive with PC-1.

A preferred way of measuring the reactivity of a PC-1 epitope with aspecific antibody of the present invention is by enzyme immunoassay(EIA) such as an enzyme-linked immunosorbent assay (ELISA), Voller, A.et al., J. Clin. Pathol., 31:507-520 (1978); Butler, J. E., Meth.Enzymol. 73:482-523 (1981); Maggio, E. (ed.), Enzyme Immunoassay, CRCPress, Boca Raton, Fla., (1980). The enzyme, when exposed to anappropriate substrate, will react with the substrate in such a manner asto produce a chemical moiety which can be detected, for example, byspectrophotometric, fluorimetric or by visual means.

Detection of PC-1 may be accomplished using any of a variety of otherimmunoassays. For example, it is possible to detect antibody binding toPC-1 through the use of a radioimmunoassay (RIA). See, for example,Weintraub, B., Prncpls. of Radioimmunoassay, 7th Training Course onRadioli-gand Assay Techniques, The Endocrine Society, (March, 1986) pp.1-5, 46-49 and 68-78; Work, T. S. et al., Laboratory Techniques andBiochemistry in Molecular Biology, North Holland Publishing Company,(New York, 1978).

Additional types of immunoassays include precipitation reactions, geldiffusion precipitation reactions, immunodiffusion assays, agglutinationassays, complement fixation assays, immunoradiometric assays, protein Aimmunoassays, and immunoelectrophoresis assays.

In a typical screening procedure wells of flat bottomed 96 well platesare coated overnight at 4° C. with 100 μl of goat anti-mouse IgGFc-specific antibodies (Cappel, Westchester, Pa.) at 2 μg/ml in PBS.After blocking plates for 1 hr with a solution of 1% BSA in PBS, platesare washed with wash buffer (0.05w Tween-20 in PBS). Hybridoma culturesupernatants (100 μl) are added and incubated for 1-2 hrs at roomtemperature. The wells are then washed with wash buffer, and 100 μl of asolution containing approximately 1 μg/ml soluble PC-1, horseradishperoxidase-conjugated anti-PC-1 Fc fragment, 2% normal mouse serum,0.256 NP-40, and 25% FCS in wash buffer are added and incubated for 2hr. After washing, the reactions are developed. Wells positive for PC-1antibodies are selected for further characterization.

Alternatively, wells of flat bottomed 96 well plates are coatedovernight at 4° C. with 100 μl of a solution containing purified PC-1 ora fragment thereof at 2 gμg/ml in PBS. After blocking plates for 1 hrwith a solution of 1% BSA in PBS, plates are washed with wash buffer(0.05% Tween-20 in PBS). Hybridoma culture supernatant (100 μl) areadded and incubated for 1-2 hrs at room temperature. The wells are thenwashed with wash buffer, and 100 Al of a solution containingapproximately 1 μg/ml horseradish peroxidase-conjugated goat anti-mousespecific antibodies (Cappel, Westchester, Pa.) 26 normal mouse serum,0.25% NP-40, and 25% FCS in wash buffer are added and incubated for 2hr. After washing, the reactions are developed. Wells positive for PC-1antibodies are selected for further characterization.

Once an antibody is isolated that reacts with the PC-1 molecule, it isdesirable to screen the hybridomas for their effectiveness inneutralizing the inhibitor activity of PC-1 for insulin receptortyrosine kinase.

To isolate a neutralizing antibody, antibodies are made using thetechniques for generating these molecules elaborated above. Thepreferred neutralizing antibody is non-immunogenic in a human anddirected against a single determinant. Following production of a panelof antibodies, the molecules are subjected to a screening process inorder to identify those molecules which meet the desired criteria (i.e.which are able to neutralize a biological activity of PC-1 either invitro or in vivo). Normally, samples of PC-1 will be exposed to thepanel of anti-PC-1 antibodies and will then be subjected to the assaysdescribed herein. Those antibodies which block the ability of PC-1 toinhibit insulin receptor tyrosine kinase activity can be selected asneutralizing antibodies.

Assaying for potential neutralizing antibodies can be accomplished in anumber of ways. In particular, the neutralizing activity of the antibodyagent of the invention against the inhibitor can be assessed bymeasuring whether the antibody agent can block the PC-1 induced decreasein insulin receptor tyrosine kinase activity as measured by insulinreceptor autophosphorylation as well as phosphorylation of the exogenoussubstrate poly(Glu-Tyr) (Example 1). Standard methods for carrying outthe assay are reported in Sbraccia et al., J. Biol. Chem. 265:4902-4907,(1990). According to this procedure, hybridoma supernatants are screenedfor their ability to effect insulin receptor autophosphorylation. In onesuch assay, the human breast carcinoma cell line MCF-7, Milazzo et al.,Cancer Rsch., 52:3924-3930 (1992), is transfected with an expressionplasmid containing PC-1 cDNA. MCF-7 cells which express PC-1 have adecreased insulin receptor tyrosine kinase activity as measured byinsulin receptor autophosphorylation. Hybridoma supernatants can beincubated with MCF-7 cells transfected to express PC-1 and assayedaccording to known methods for measuring autophosphorylation of theinsulin receptor. In a preferred method, transfected MCF-7 cells arecoated onto 10 cm. plates in 100 μl of media and cultured overnight at37° C. in a humidified atmosphere. The wells are lightly tapped toremove the culture media and 100 μl of hybridoma supernatant is added tothe wells. The wells are incubated for an additional 30 minutes in thepresence of insulin and the excess hybridoma media is decanted. Next,the MCF-7 cells are lysed to solubilize the insulin receptors. Lysis isconducted in the presence of phosphatase inhibitors in a bufferconsisting of 150 mM NaCl containing 50 mM HEPES, 0.5% TRITON® X-100,0.01% thimerosal, 30 KIU/ml aprotinin, 1 mM4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 50CM leupeptin,and 2 mM sodium orthovanadate, pH 7.5. The lysates are then screened forthe presence of phosphotyrosine using a standard ELISA procedure.

In the subsequent ELISA, insulin receptors are captured on a 96 wellplate precoated with anti-insulin receptor monoclonal antibodies. Theplates are then incubated with biotinylated 4G10 (anti-phosphotyrosineantibody, Upstate Biotechnology, Inc, Lake Placid, NY). After incubationfor 2 hours at room temperature, the plate is washed free of excess 4G10and incubated with 100 μl horseradish peroxidase conjugated streptavidin(Zymed laboratories, S. San Francisco, Calif.). The plates are incubatedfor 30 minutes and excess streptavidin washed away. Substrate(tetraethyl benzidine) is then added and the reaction developed forcolor. Absorbance is read at 450 nm.

Since the MCF-7 cells are transfected with a cDNA clone containing thefull length PC-1 these cells are expected to overexpress the membraneglycoprotein. Normal insulin receptor autophosphorylation is decreasedin these cells. Hybridoma supernatants which restore insulin receptortyrosine kinase autophosphorylation are selected for furthercharacterization.

For measuring the potential preventative, suppressive or therapeuticbenefit of the antibody agents of the present invention certain in vitroand, in vivo clinical outcomes can be assessed. Several additionalcellular assays exist that are useful in the context of the presentinvention. These include the glucose uptake assay utilizing the mouse3T3-L1 adipocytes Cheatham et al., Mol. Cell, Bio., 14(7):4902-4911(1994). According to this procedure, potential antibody agents areselected according to their ability to effect insulin-stimulated glucoseuptake. The antibody agents of the present invention can also be used toevaluate the ability of insulin to stimulate mitogenesis by assayseasily recognized by one of ordinary skill in the art.

Next, the potential antibody agents are screened in a number ofdifferent in vivo situations. One skilled in the art will recognize thatthe agents can be evaluated according to their effects on sensitivity toglucose in appropriate murine models. Several animal model systemstesting the therapeutic benefit of the antibody agents of the inventionexist. These include models of insulin resistance such as the male obeseWistar Diabetic Fatty rat model of NIDDM (Greene, S., Obesity Re,, 2:432(1994)). The insulin resistant obese diabetic Zucker rat is an animalmodel of human type 2 NIDDM, Terrettaz and Jeanrenaud, Endocrinology,112:1346-1351 (1983); Haring, H. and Obermaier-Kusser, B.,Diabetes/Metabolism Reviews, 5:431 (1989).

Additionally, agents can be screened according to their ability toeffect any of several in vivo parameters. The spontaneously obese rhesusmonkey is a suitable model system for these studies Bodkin et al., Amer.J. Physiol., 256(5 pt.2):E676-681 (1989). According to this model plasmalevels of insulin and glucose can be determined, Hansen & Bodkin,Diabetologia, 29:713-719 (1986) in the fasting state and followingintravenous injection of glucose.

7. Selecting Appropriate Antibodies

Once antibodies of the desired specificity are generated, they may beused to identify and select other antibodies having the same orcross-reactive epitope specificity. For example, a new antibody istested by measuring its ability to inhibit the binding of an antibody ofknown specificity to its epitope. Various competitive binding assaysknown in the art can be used.

The isotype of the antibody can be selected during hybridoma productionor by appropriate recombinant methods well-known in the art to achieve adesired effector function mediated by the Fc portion of theimmunoglobulin heavy chain. For example, certain isotypes, such asIgG2a, have superior activity in antibody-dependent cellularcytotoxicity. Likewise, certain isotypes, such as IgG2a, are morereadily eliminated from the circulation through Fc receptors on cells ofthe reticuloendothelial system and are therefore more efficient atremoving an undesired antigen or target cell from sites of activedisease. Accordingly, depending on the intended use, a particularantibody isotype may be preferable to others, as can be readilyascertained by one of ordinary skill in the art without undueexperimentation.

To identify a hybridoma producing an antibody of a particular isotype,or to switch an isotype of an antibody, the hybridoma supernatants maybe screened for production of PC-1 specific mAbs using an ELISA whichtests for the immunoglobulin isotype. What follows is an example of amethod for selecting a desired isotype switch from IgG1 to IgG2a.Hybridoma cells are grown in the logarithmic phase for a 2-3 week periodprior and then subjected to negative selection using antibody-coatedmagnetic beads. Super paramagnetic iron oxide particles coated with agoat anti-mouse antibody preparation including all IgG isotype classes(BIOMAG® beads purchased from Advanced Magnetics, Inc.) may be used. Forswitching an isotype from IgG1 to IgG2a, it is necessary to block theIgG2a binding sites on the antibody-coated beads by incubating withimmunoglobulins (of irrelevant specificity) having the IgG2a isotype.About 10⁸ hybridoma cells expressing a variety of isotypes are incubatedwith such IgG2a-blocked beads. Cells expressing IgG1, IgG2b and IgG3isotypes bind and are removed magnetically from the population. Such anegative selection step is preferably repeated several times.

The remaining cell population, depleted of IgG1, IgG2b and IgG3 bearingcells, and conversely enriched for IgG2a-bearing cells, is plated inmicroplates at a cell density of about 1000 cells/well. Usingcommercially available anti-isotype reagents in an ELISA assay, thewells are screened for IgG2a production; positive clones are replated at0.3 cells/well followed by another round of screening and re-plating.Using such an approach, approximately 1-5 of 10⁷ cells which haveswitched isotype are optimally selected. Cells which have switched fromIgM to IgG can be selected using a similar approach with the appropriateantibody-coated beads.

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson and Pollard, Anal.Biochem., 107:220 (1980). Antibodies with an affinity for PC-1 of aboutin the range of 10⁸ M-1 and greater are useful with in the context ofthe present invention.

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, DMEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxyapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of DNA encoding the antibody includeSkerra et al., Curr. Opinion in Immunol., 5:256-262 (1993); andPluckthun, Immunol. Revs., 130:151-188 (1992).

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences, Morrison et al., Proc. Nat. Acad. Sci.,81:6851 (1984), or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpoly-peptide. In that manner, "chimeric" or "hybrid" antibodies areprepared that have the binding specificity of an anti-PC-1 monoclonalantibody herein.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for a PC-1 andanother antigen-combining site having specificity for a differentantigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include 2-iminothiol andmethyl-4-mercaptobutyrimidate.

8. Humanized antibodies

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as "import" residues, whichare typically taken from an "import" variable domain. Humanization canbe essentially performed following the method of Winter and co-workers,Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); and Verhoeyen et al., Science, 239:1534-1536 (1988),by substituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such "humanized" antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called "best-fit" method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody, Sims et al., J.Immunol., 151:2296 (1993); and Chothia et al., J. Mol. Biol., 196:901(1987). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies, Carter et al., Proc, Natl. Acad. Sci.USA, 89:4285 (1992); and Presta et al., J. Immuno., 151:2623 (1993).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

9. Human antibodies

Human monoclonal antibodies can be made by the hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described, for example, by Kozbor,J. Immunol., 133:3001 (1984); Brodeur, et al., Monoclonal Antibody Prod.Techniques and Applns., pp.51-63, Marcel Dekker, Inc., N.Y., (1987); andBoerner et al., J. Immunol., 147:86-95 (1991).

It is now possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); and Bruggermann et al., Year in Immuno., 7:33(1993).

Alternatively, phage display technology, McCafferty et al., Nature,348:552-553 (1990) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology, 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol., 222:581-597 (1991), or Griffith et al., EMBO J.,12:725-734 (1993). In a natural immune response, antibody genesaccumulate mutations at a high rate (somatic hypermutation). Some of thechanges introduced will confer higher affinity, and B cells displayinghigh-affinity surface immunoglobulin are preferentially replicated anddifferentiated during subsequent antigen challenge. This natural processcan be mimicked by employing the technique known as "chain shuffling"(Marks et al. Bio/Technol., 10:779-783 (1992). In this method, theaffinity of "primary" human antibodies obtained by phage display can beimproved by sequentially replacing the heavy and light chain V regiongenes with repertoires of naturally occurring variants (repertoires) ofV domain genes obtained from unimmunized donors. This technique allowsthe production of antibodies and antibody fragments with affinities inthe nM range. A strategy for making very large phage antibodyrepertoires has been described by Waterhouse et al., Nucl. Acids Res.,21:2265-2266 (1993).

Gene shuffling can also be used to derive human antibodies from rodentantibodies, where the human antibody has similar affinities andspecificities to the starting rodent antibody. According to this method,which is also referred to as "epitope imprinting", the heavy or lightchain V domain gene of rodent antibodies obtained by phage displaytechnique is replaced with a repertoire of human V domain genes,creating rodent-human chimeras. Selection on antigen results inisolation of human variable capable of restoring a functionalantigen-binding site, i.e. the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT WO 93/06213,published Apr. 1, 1993). Unlike traditional humanization of rodentantibodies by CDR grafting, this technique provides completely humanantibodies, which have no framework or CDR residues of rodent origin.

10. Uses for Antibody Agents

Anti-PC-1 antibodies, especially neutralizing antibodies, are useful indiagnostic assays for the presence of PC-1 overexpression, e.g., itsproduction in specific cells, tissues, or serum. For example, PC-1antibodies can be used for a method for detecting or measuring theamount of an insulin receptor tyrosine kinase inhibitor in a samplecomprising the steps of(a) contacting a sample with a first anti-PC-1antibody under conditions which allow immunospecific binding to occur(b) contacting the sample with a second anti-PC-1 antibody underconditions which allow immunospecific binding to occur; and(c) detectingor measuring any immunospecific binding that occurs of a component ofthe sample with both the first and the second anti-inhibitor antibodies,in which immunospecific binding of a component of the sample with saidfirst and second antibodies indicates the presence or amount of theinhibitor in the sample.

The invention also includes a method for determining the overexpressionof an insulin receptor tyrosine kinase inhibitor in a sample comprisingthe steps of: (a) measuring the total amount of an insulin receptortyrosine kinase inhibitor according to the method described supra; andcomparing the amount determined in step (a) to an amount of insulinreceptor tyrosine kinase inhibitor present in a standard sample, anincreased level in the amount of step (a) being indicative of anoverexpression of the insulin receptor tyrosine kinase inhibitor.

The invention thus provides a method for detecting the presence or theonset of insulin resistance or non-insulin dependent diabetes mellitus,or other disease or disorder involving inappropriate glucose metabolismcomprising the steps of; measuring the amount of an insulin receptortyrosine kinase inhibitor in a sample according to the method describedsupra and comparing the amount of the insulin receptor tyrosine kinaseinhibitor in the sample to the amount of insulin receptor tyrosinekinase inhibitor in a standard sample, an overabundance of insulinreceptor tyrosine kinase inhibitor in the sample being indicative of thedisorder.

11. Assays Formats and Kits

Many different assays and assay formats can be used to detect the amountof PC-1 in a sample relative to a control sample. These formats, in turnare useful in the diagnostic assays of the present invention. Thediagnostic assays are useful in the identification of diseases anddisorders involving increased or abnormal levels of PC-1.

Any procedure known in the art for the measurement of soluble analytescan be used in the practice of the instant invention. Such proceduresinclude but are not limited to competitive and non-competitive assaysystems using techniques such as radioimmunoassay, enzyme immunoassays(EIA), preferably the enzyme linked immunosorbent assay (ELISA),"sandwich" immunoassays, precipitin reactions, gel diffusion reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays, and immunoelectrophoresis assays, to name but a few. Forexamples of preferred immunoassay methods, see U.S. Pat. No. 4,845,026(Jul. 4, 1989) and U.S. Pat. No. 5,006,459 (Apr. 9, 1991).

In one embodiment, one or more of the antibodies used in an assay tobind a PC-1 molecule according to the invention are labeled; in anotherembodiment, a first is unlabeled, and a labeled, second antibody is usedto detect the PC-1 bound to the first antibody. A further methodincludes the assay where for instance a rat IgG monoclonal antibody isused to detect or measure PC-1(antigen) in a sample by binding theretoLabeled goat anti-rat immunoglobulin can then be used to detect thebound monoclonal antibody.

In a preferred embodiment, polyclonal and/or monoclonal antibodies canbe used in sandwich immunoassays according to the invention. In aparticular embodiment, a first antibody is not used and the PC-1 isbound directly to a solid support and a second binding partner which isan antibody or antibody fragment or derivative is used in the detection.

In an EIA, the enzymes which can be used to detectably label an antibodyinclude, but are not limited to, horseradish peroxidase, alkalinephosphatase, glucose-6-phosphate dehydrogenase, malate dehydrogenase,staphylococcal nuclease, Δ-V-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphateisomerase, asparaginase, glucose oxidase, X-galactosidase, ribonuclease,urease, catalase, glucoamylase and acetylcholinesterase.

It is also possible to label the detection antibody with a fluorescentcompound. When the fluorescently labeled antibody is exposed to light ofthe proper wavelength, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labellingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The detecting antibody can also be detectably labeled using fluorescenceemitting metals such as ¹⁵² Eu, or others of the lanthanide series.These metals can be attached to the antibody using such metal chelatinggroups as diethylenetriaminepentaacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester. Likewise, a bioluminescent compound may be used to labelthe antibody. Bioluminescence is a type of chemiluminescence found inbiological systems in which a catalytic protein increases the efficiencyof the chemiluminescent reaction. The presence of a bioluminescentprotein is determined by detecting the presence of luminescence.Important bioluminescent compounds for purposes of labeling areluciferin, luciferase and aequorin.

Any other label known in the art may be used, e.g., a radionuclide, etc.

In the assays of the present invention, an antigen such as PC-1, or anantibody is preferably bound to a solid phase support or carrier. By"solid phase support or carrier" is intended any support capable ofbinding an antigen or antibodies. Well-known supports, or carriers,include glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amyloses, natural and modified celluloses, polyacrylamide, agaroses, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

In a preferred embodiment, an antibody-antigen-antibody sandwichimmunoassay is done, i.e., antigen is detected or measured by a methodcomprising binding of a first antibody to the antigen, and binding of asecond antibody to the antigen, and detecting or measuring antigenimmunospecifically bound by both the first and second antibody. In aspecific embodiment, the first and second antibodies are monoclonalantibodies. In this embodiment, if the antigen does not containrepetitive epitopes recognized by the monoclonal antibody, the secondmonoclonal antibody must bind to a site different from that of the firstantibody (as reflected e.g., by the lack of competitive inhibitionbetween the two antibodies for binding to the antigen). In anotherspecific embodiment, the first or second antibody is a polyclonalantibody. In yet another specific embodiment, both the first and secondantibodies are polyclonal antibodies.

In a preferred embodiment, a "forward" sandwich enzyme immunoassay isused, as described schematically below. An antibody (capture antibody,Ab1) directed against the PC-1 is attached to a solid phase matrix,preferably a microplate. The sample is brought in contact with theAb1-coated matrix such that any PC-1 in the sample to which Ab1 isspecific binds to the solid-phase Ab1. Unbound sample components areremoved by washing. An enzyme-conjugated second antibody (detectionantibody, Ab2) directed against a second epitope of the antigen binds tothe antigen captured by Ab1 and completes the sandwich. After removal ofunbound Ab2 by washing, a chromogenic substrate for the enzyme is added,and a colored product is formed in proportion to the amount of enzymepresent in the sandwich, which reflects the amount of antigen in thesample. The reaction is terminated by addition of stop solution. Thecolor is measured as absorbance at an appropriate wavelength using aspectrophotometer. A standard curve is prepared from knownconcentrations of the antigen, from which unknown sample values can bedetermined.

Other types of "sandwich" assays are the so-called "simultaneous" and"reverse" assays. A simultaneous assay involves a single incubation stepas the antibody bound to the solid support and labeled antibody are bothadded to the sample being tested at the same time. After the incubationis completed, the solid support is washed to remove the residue of fluidsample and uncomplexed labeled antibody. The presence of labeledantibody associated with the solid support is then determined as itwould be in a conventional "forward" sandwich assay.

In the "reverse" assay, stepwise addition first of a solution of labeledantibody to the fluid sample followed by the addition of unlabeledantibody bound to a solid support after a suitable incubation period isutilized. After a second incubation, the solid phase is washed inconventional fashion to free it of the residue of the sample beingtested and the solution of unreacted labeled antibody. The determinationof labeled antibody associated with a solid support is then determinedas in the "simultaneous" and "forward" assays.

Kits comprising one or more containers or vials containing componentsfor carrying out the assays of the present invention are also within thescope of the invention. For instance, such a kit can comprise anantibody or antibodies, preferably a pair of antibodies to the PC-1antigen which preferably do not compete for the same binding site on theantigen. In a specific embodiment, PC-1 may be pre-adsorbed to the solidphase matrix. The kit preferably contains the other necessary washingreagents well-known in the art. For EIA, the kit contains thechromogenic substrate as well as a reagent for stopping the enzymaticreaction when color development has occurred. The substrate included inthe kit is one appropriate for the enzyme conjugated to one of theantibody preparations. These are well-known in the art, and some areexemplified below. The kit can optionally also comprise a PC-1 standard;i.e., an amount of purified PC-1 corresponding to a normal amount ofPC-1 in a standard sample.

In a specific embodiment, a kit of the invention comprises in one ormore containers: (1) a solid phase carrier, such as a microtiter platecoated with a first antibody; (2) a detectably labeled second antibody;(3) a standard sample of the PC-1 molecule recognized by the first andsecond binding partners.

12. Additional Antibody Agent Uses

The agent herein may also be used as an affinity purification agent. Inthis process, the antibodies against the inhibitor are immobilized on asuitable column such as a Sephadex column by any conventional technique.The sample containing the inhibitor to be separated is placed on thecolumn and the column is washed with a suitable solvent that will letall molecules pass except the inhibitor, which adheres to the column.Then the column is washed with a suitable solvent, such as glycinebuffer, pH 5.0, to elute the Inhibitor from the column.

The antibodies herein are also useful in a receptor binding orradioreceptor assay in a conventional manner, as by incubating a mixtureof radiolabeled inhibitor (e.g., radioiodinated inhibitor) and theantibody with cells containing the insulin receptor, and determining ifthe antibody blocks binding of the labeled inhibitor to the receptor.

13. Soluble Receptors or Fragments Thereof

In another preferred aspect "agent" is meant to include a portion of aninsulin receptor that binds the PC-1 molecule or interferes with thePC-1 interaction with the membrane associated insulin receptor and, byvirtue of binding or interfering, neutralizes the effect of the PC-1 onthe tyrosine kinase activity of the membrane associated receptor. Thesoluble receptor may be a portion of the membrane associated insulinreceptor such as a peptide or fragment thereof. According to this aspectof the invention care must be taken to select a soluble insulin receptorthat does not bind insulin in vivo but has the desirable quality ofpreventing the interaction of the PC-1 molecule with the membraneassociated insulin receptor. Ideally, the soluble receptor fragment willprevent or block the PC-1 dependant inhibition of the membrane boundinsulin receptor tyrosine kinase activity.

Such soluble receptor molecules are based in whole or in part on theinsulin receptor primary structure. The primary structure of the insulinreceptor as well as the nucleic acid encoding the receptor can be foundin U.S. Pat. No. 4,761,371 issued Aug. 2, 1988, by Bell et al.

Soluble insulin receptor constructs can therefore be devised which lackall or a portion of the transmembrane region of the insulin receptor. Tothis end an expression vector can be constructed to encode an insulinreceptor molecule which lacks the carboxyl-terminal transmembraneregion. In one embodiment the fragment retains the desirable property ofbinding PC-1 in vivo. By binding PC-1, the soluble receptor fragmentprevents the interaction of the PC-1 with the membrane associatedinsulin receptor.

Portions of the insulin receptor molecule which are important for therecognition of insulin can be determined by those skilled in the art.Additionally, site-directed mutagenesis such as those techniquesdescribed by Kunkel et al., Methods of Enzymol. 154:367-382 (1987), aswell as alanine scanning mutagenesis as described in Cunningham andWells, (1989) Science, 244:1081-1085 can be employed, as well as otherroutine techniques, to elucidate the portions of the insulin receptorcritical to insulin binding. A soluble molecule based on the primarystructure of the insulin receptor that does not bind insulin but whichhas the desirable quality of preventing the interaction of the PC-1 withthe membrane bound insulin receptor can thus be devised.

To this end an expression vector can be constructed to encode a solublemolecule based on the primary sequence of insulin receptor. After thesequence encoding the portion of the insulin receptor having thedesirable properties is prepared, expression can be obtained usingtechniques available to those of skill in the art.

Potential candidates for the soluble molecule can be screened accordingto their ability to inhibit PC-1 dependent inhibition of insulinreceptor tyrosine kinase activity. Assays which monitor theautophosphorylation of the insulin receptor, as well as phosphorylationof exogenous substrates, such as those described herein can be used toselect the appropriate soluble receptor molecule.

14. Therapeutic Use of Agents

The preclinical and clinical therapeutic use of the present invention inthe treatment of diseases or disorders associated with overexpression ofPC-1 will be best accomplished by those of skill, employing acceptedprinciples of diagnosis and treatment. Such principles are known in theart, and are set forth, for example, in Braunwald et al., eds.,Harrison's Prncpls. of Intnl. Med., 11th Ed., McGraw-Hill, N.Y. (1987).

The agents of the present invention provide distinct advantages in thetreatment of a disease or disorder involving inappropriate PC-1expression. Prior to the instant invention, therapeutic options fortreatment of improper glucose metabolism as a result of insulinresistance included: (a) no treatment with possible spontaneousresolution; (b) treatment with rigorous dietary modification to controlpostprandial glucose levels c) costly insulin supplementation therapy.The therapeutic methods of the instant invention have the distinctadvantage over the prior art methods of treating a disorder associatedwith improper glucose metabolism as a result of overexpression ofmembrane glycoprotein PC-1. The methods provide for specific,inexpensive intervention at the probable pathogenic origin of theimbalance.

For therapeutic applications, the agents may be administered to amammal, preferably a patient, in a pharmaceutically acceptable dosageform, including those that may be administered to a patientintravenously as a bolus or by continuous infusion over a period ofminutes, hours, days, weeks, or months, intramuscularly, subcutaneously,intra-articularly, intrasynovially, intrathecally, or periostally, or byoral, topical, or inhalation routes.

The most effective mode of administration and dosage regimen of agentwill depend on the type of disease to be treated, the severity andcourse of the disease, whether the agents are administered forprophylactic or therapeutic purposes, previous therapy, the patient'sclinical history and response to the agents such as antibodies, and thediscretion of the attending physician. The agent is suitablyadministered to the patient at one time or over a series of treatments.

A dose of agent may be administered to the patient, whether via, e.g.,one or more single administrations, continuous infusion, or bolusinjection. For example, an initial dose of the agent is administered tothe patient by injection or infusion. For repeated administrations overseveral days or longer, depending on the condition, the treatment isrepeated until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. According to anotherembodiment of the invention, the effectiveness of the agent may beimproved by administering the agent serially or in combination withanother agent that is effective for this purpose.

Several clinical parameters can be followed over the course of treatmentincluding monitored clinical improvement in 1) glycemic control andconsequences thereof including for instance, normalization of serumlipids, and reduced risk for late vascular complication; 2) stabilizedglycemic control, for instance, reduced brittleness and reduced risk foracute decompensation; and 3) reduced incidence of diabetic ketoacidosis.

An amount of agent capable of preventing PC-1 dependent inhibition ofinsulin receptor tyrosine kinase activity when provided to a patient isa "therapeutically effective" amount, which is generally about 0.01 to100 mg/kg body weight/day depending on the factors noted above. Thedosage of an antibody agent may be given by an intravenously injectabledose in the range of about 0.01 to 25 mg/kg body weight/day.

Pharmaceutical Compositions of the Invention

The agents of the present invention including antibodies, and solublereceptor fragments are well suited for the preparation of pharmaceuticalcompositions. The pharmaceutical compositions of the invention may beadministered to any animal which may experience the beneficial effectsof the compositions of the invention. Foremost among such animals arehumans, although the invention is not intended to be so limited.

In addition to the agent itself pharmacologically active, pharmaceuticalcompositions preferably contain suitable pharmaceutically acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically.

Suitable formulations for parenteral administration include aqueoussolutions of the peptides in water-soluble form, for example,water-soluble salts. In addition, suspensions of the proteins orpeptides as appropriate oily injection suspensions may be administered.Suitable lipophilic solvents or vehicles include fatty oils, forexample, sesame oil, or synthetic fatty acid esters, for example, ethyloleate or triglycerides. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension include, forexample, sodium carboxy-methyl cellulose, sorbitol, and/or dextran.Optionally, the suspension may also contain stabilizers. The agents ofthe invention are preferably formulated in purified form substantiallyfree of aggregates and other protein materials.

The compositions are formulated using conventional pharmaceuticallyacceptable parenteral vehicles for administration by injection. Thesevehicles are nontoxic and therapeutic, and a number of formulations areset forth in Remington's Pharmaceutical Sciences, 16th ed., MackPublishing Co., (1980). Non-limiting examples of excipients are water,saline, Ringer's solution, dextrose solution and Hank's balanced saltsolution. Formulations according to the invention may also contain minoramounts of additives such as substances that maintain isotonicity,physiological PH, and stability. Such dosage forms encompasspharmaceutically acceptable carriers that are inherently nontoxic andnontherapeutic. Examples of such carriers include ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts, or electrolytes such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, and polyethyleneglycol.

Adjuvants for topical or gel-based forms of agents includepolysaccharides such as sodium carboxymethylcellulose ormethylcellulose, polyvinylpyrrolidone, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol,and wood wax alcohols.

For all administrations, conventional depot forms are suitably used.Such forms include, for example, microcapsules, nano-capsules,liposomes, plasters, inhalation forms, nose sprays, and sublingualtablets.

The examples that follow demonstrate the present invention. The methodsemployed herein are exemplary only. It will be apparent that variousdepartures from and modifications of these techniques may be made inlight of the present specification and the ordinary level of skill inthe art without departing from the spirit and scope of the invention.All literature and patent citations in the examples are expresslyincorporated by reference.

EXAMPLES

1. General Methods

1.1 Autophosphorylation of Exogenous Substrate

Insulin-receptor autophosphorylation and insulin-receptor tyrosinekinase activity employing the artificial substrate poly(Glu-Tyr) werecarried out according to standard methods. Sbraccia et al., J. Biol.Chem. 265:4902-907 (1990). 1 ng insulin receptor was incubated in 25 μlbuffer containing 2 mM MnCl₂, 10 mM MgCl₂, 50 mM HEPES (pH 7.6), 150 mMNaCl, 0.1% TRITON® X-100, and 1% BSA in the presence or absence of 100nM insulin for 1 h at 20° C. A 5 μl mixture of 1 mg/ml poly(Glu-Tyr) and10 μM[³² P]ATP was added for 1 h at 20° C. ³² P incorporation was thenmeasured by trichloroacetic acid precipitability of filter paper.

Example 2 Insulin Receptor Antagonists in Fibroblasts from an NIDDMPatient

Methods

Insulin Receptor Autophosphorylation

Fibroblasts from patients as well as sex and age matched controls wereobtained by forearm skin biopsy and grown under standard conditions inDulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetalbovine serum at 37° C. in a humidified atmosphere (Tissue Culture:Methods and Applns., Kruse P. F. Jr. et al., Eds., N.Y., Academic(1973). At the point of assay 1 nM insulin was added to culturescontaining 1×10⁶ fibroblast cells for 2 min. The cells were then thricewashed with PBS at 4° C., and scraped into 1 ml of buffer containing 50mM HEPES (pH 7.6), 150 mM NaCl, 1 mM p-methyl sulfonyl fluoride (PMSF)and 2 mM sodium orthovanadate. Cells were pelleted, and solubilized inthe same buffer without NaCl but containing 1% TRITON® X-100. Thelysates were immunoabsorbed with a-IR3 (a monoclonal antibody specificto the IGF-I receptor). The depleted lysates were thenimmunoprecipitated with an anti-insulin receptor antiserum as describedin Forsayeth et al., Diabetes, 35:837-846 (1986) and immunoprecipitatessubjected to SDS-PAGE in an 8-16% gradient gel (NOVEX) under reducingconditions. Proteins were transferred to nitrocellulose andimmunoblotted with an antibody to phosphotyrosine (UpstateBiotechnology, Inc.)

Results

To confirm the presence of an inhibitor to insulin receptor tyrosinekinase activity in intact fibroblasts, in vivo tyrosine kinase activitywas assessed by insulin receptor β subunit autophosphorylation asdescribed supra. In control fibroblasts, 1 nM insulin stimulated insulinreceptor β-subunit autophosphorylation (FIG. 2). Maximal effects wereseen at 10 to 100 nM Insulin (FIG. 2). The fibroblasts from the patientwith insulin resistance required significantly more insulin to stimulateautophosphorylation of the insulin receptor in vivo.

Example 3 Purification of the Insulin Receptor Tyrosine Kinase InhibitorPC-1

Fibroblasts (5×10⁸) from an individual showing marked inhibition ofinsulin receptor autophosphorylation in vivo were scraped and washed.Cell pellets were solubilized in 50 mM HEPES (pH 7.6), 1 mM PMSF, 2 mMsodium orthovanadate, and 1% TRITONμ X-100. Solubilized lysates wereapplied to an anti-insulin receptor antibody affinity column, Sbraccia,et al., (1991); Diabetes, 40:295-299; and Maddux, B. A., J. Clin. End.Metab. 77:73-79 (1993). The passthrough (depleted of insulin receptors)was then applied to a wheat germ agglutinin agarose column (EY Labs).After washing, the bound glycoproteins were eluted with 0.3 MN-acetyl-D-glucosamine in 50 mM HEPES (pH 7.6), 150 mM NaCl, 0.01%Tween-20 and 1 mM PMSF. The glycoproteins were desalted using aCentricon 100 filter (Amicon Corp) and applied to a 1 ml ATP agarosecolumn (Sigma). After washing, proteins were eluted with a NaCl stepgradient. Both the wheat germ agglutinin eluate and the 1 M NaCl eluatewere subjected to SDS-PAGE followed by silver staining.

Cell Free Insulin Receptor Tryosine Kinase Assay

The purification of PC-1 was followed by the cell free insulin receptortyrosine kinase assay described below.

1 ng insulin receptor was incubated in 25 μL buffer containing 2 mmMnCl₂, 10 mM MgCl₂, 50 mM HEPES (pH 7.6), 150 mM NaCl, 0.1% TRITON®X-100, and 0.1% BSA in the presence or absence of 100 nM insulin for 1 hat 20° C. A 5 μL mixture of 1 mg/mL poly(Glu-Tyr) and 10 μM [³² P]ATPwas added for 1 h at 20° C. ³² p incorporation was then measured bytrichloroacetic acid precipitability on filter paper. In the initialstudies, 5 μL wheat germ purified extracts containing 0.3 ng insulinreceptor immunoreactivity were added to ing purified human insulinreceptor from transfected 3T3/HIR mouse fibroblasts expressing 10⁶ humaninsulin receptor/cell Whittaker et al., Proc. Natl. Acad. Sci. USA,84:5237-5241 (1987). For subsequent studies with extracts depleted ofreceptor, 5 μL of extract containing 200 ng inhibitor protein were addedto purified receptors.

Results

The inhibitor was purified using a cell free insulin receptor tyrosinekinase assay to follow the purification process. Solubilized celllysates were depleted of insulin receptors, and the inhibitor purifiedto apparent homogeneity by wheat germ affinity chromatography followedby ATP-agarose chromatography.

The inhibitor eluted from the ATP-agarose column as two bands withrelative molecular weights of 130 and 260 kDa (FIG. 3). The ATP-agaroseeluate was >100 fold more potent as an inhibitor of insulin receptortyrosine kinase activity than the wheat germ eluate. Purification fromthe cell homogenate was >1000 fold as determined by western blotting andenzyme activity.

The 130,000 kDa band was eluted and cleaved with cyanogen bromide; theresulting fragments were separated on a thin SDS gradient gel (0.5 mm)to increase electroblotting transfer efficiency. Bands at 14 and 21 kDawere sequenced (data not shown), and found by Automated Edmandegradation to be identical to the corresponding cyanogen bromidepeptides of the membrane glycoprotein, PC-1, Buckley et al., J. Biol.Chem., 265:17506-17511 (1990); and Funakoshi, et al., Arch. Biochem.Biophys. 295:180-187 (1992).

Example 4 Western Blot Analysis of PC-1 Protein Content in Fibroblasts

Western blotting of lysates from a patent with insulin resistance andNIDDM (MW) cells revealed that there was a 5-10 fold increase in boththe 130 kDA (monomer) and 260 kDa (dimer) forms of PC-1 when compared tocontrol cells.

Methods

One μg/lane of inhibitor was electrophoresed in 12% SDS-PAGE usingnon-reducing sample buffer along with the appropriate controls andstandards. The glycoprotein bands were then transferred to anitrocellulose solid support membrane via semi-dry electrophoretictransfer. The membranes containing transferred glycoproteins wereblocked with 1% non-fat dry milk proteins for 2 h or other blockingreagents such as BSA at about 2% and gelatin at about 0.3%. Thenitrocellulose membranes were then reacted with polyclonal antibodies toPC-1. The membranes were then washed with buffer (PBS-Tween) 4 times forabout 5-10 minutes each. The membranes were then reacted withHorseradish peroxidase labelled anti-murine antibodies for 2 h. Themembranes were again washed and subsequently developed with substrate tovisualize the glycoprotein bands.

Results

Western blotting of lysates from cells of a patient with insulinresistance and non-insulin dependent diabetes mellitus revealed thatthere was a 5-10 fold increase in both the 130 kDA (monomer) and 260 kDa(dimer) forms of PC-1 when compared to control cells. The results aredemonstrated in FIG. 4A.

Example 5 Northern Blot Analysis of PC-1 mRNA expression

Methods

Fibroblast cells were grown to confluency and 10 μg polyA⁺ mRNA wasprepared by proteinase K digestion (Hartmann et al., Endocrinology,127:2038-2049 (1990). 10 μg poly(A)+ was subjected to 1% agaroseformaldehyde electrophoresis, transferred to nitrocellulose, and probedwith cDNA's to human PC-1, Warrem et al., An. Intern. Med., 113:909-915(1990), or B-actin (Hartmann, supra)

Results

In mRNA from MW, there was 5-10 fold increase in the major mRNA speciesfor PC-1 (FIG. 4b); B-actin mRNA was unchanged (FIG. 4c).

Example 6 PC-1 Studies in Dermal Fibroblasts from NIDDM Patients andControls

Methods

Assay for PC-1 activity: Fibroblasts from controls and NIDDM patientswere grown to confluency, washed as described, and solubilized in 150 mMNaCl, 1% Triton X-100, 1 mM PMSF, and 20 mM imidazole (pH 7.8) for 1 hat 4° C. Supernatants containing 0.05-3 μg protein were then incubatedwith 9 nmol [³⁵ S]3'-phosphoadenosine, 5'-phosphosulfate (NEN) in thepresence of 0.1 μmol MgCl₂ in 20 μl buffer (0.1 M 2amino-2-methyl-1-propanol-HCl, pH 9.4) (Sigma) for 30 min at 37° C. 25μl 0.1 M sodium acetate (pH 5.5) was added and samples were boiled 1min. Then 0.5 ml activated charcoal (40 mg charcoal/ml in 20 mM sodiumsulfate) was added. After 10 min on ice, tubes were centrifuged, andsupernatants counted.

Western Blot analysis: Fibroblasts from 3 controls (C₁,C₂,C₃) and 4NIDDM patients (D₁,D₂,D₃,D₄) were analyzed for PC-1 content by westernblot analysis as described above.

Cells were analyzed for insulin receptor B-subunit autophosphorylationas described above.

Results

Since PC-1 has enzyme activity that hydrolyses phosphosulfate bonds, itcan be measured using the synthetic substrate 31'-phosphoadenosine,5'-phosphosulfate (PAPS), Yoshida et al., J. Biochem., 93:1641-1648(1983). PAPS hydrolysis in fibroblasts from MW was 417 nmoles/mgprotein/min, a value 10 fold greater than that of 11 control subjects,42±7 (mean±SEM, range 22-76) (FIG. 5a). When extracts of Patientfibroblasts and the purified PC-1 protein were treated with an antiserumto PC-1, >900 of both PC-1 activity, and inhibition of insulin receptortyrosine kinase activity were removed.

Fibroblasts from 9 additional patients with typical NIDDM (4 males and 5females) were studied (FIG. 5A). The average age was 51 years (mean),body mass index 28.6±2.7 kg.M², fasting glucose 223±22 mg/dl; andfasting insulin 29±9 μU/ml. PC-1 activity in the patient fibroblasts was128±25 nmoles/mg protein/min (mean±SEM); fibroblasts from 2 of the 9patients gave PC-1 values in the normal range. In 4 patients with highPC-1 activity, we measured fibroblast PC-1 content by Western blottingas described supra. PC-1 was increased when compared to controls (FIG.5B). PC-1 content was not increased in the 2 NIDDM patients with lowPC-1 activity. In fibroblasts from 2 of the patients with high PC-1activity (D2, D4), insulin receptor tyrosine kinase activity was alsodecreased (FIG. 5C).

Example 6 Effect of Overexpression on MCF-7 Cells

Methods

Human MCF-7 cells were transfected both with an expression vector, Suvaet al., Gene, 77:95-105 (1989), containing the coding sequence of humanPC-1, under the control of the cytomegalovirus promoter, and pRK-neo, aselectable marker for neomycin resistance. For controls, MCF-7 cellswere transfected with pRK-neo alone. Confluent cells were incubated with20 pM of ¹²⁵ I-insulin in the presence or absence of unlabelled insulin,Milazzo et al., Cancer Rsch., 52:3924-3930 (1992), for 18 h at 4° C.Binding to plates was carried out as described in Milazzo et al. supra.

Cells were grown to .sup.˜ 90% confluency, washed, and media was changedto DMEH-21 with 0.1% BSA. Cells were incubated with insulin for 2 min at37° C., and then solubilized as in Example 2. Lysates were Westernblotted as in Example 2, but without prior immunoprecipitation. Shown inFIG. 6B is a representative experiment with pRK-neo transfected and PC-1transfected MCF-7 cells.

MCF-7 cells were prepared as described above and preincubated 16 h withinsulin plus 5 nM a-IR3 (an antibody to the IGF-I receptor) to block theIGF-I receptor, and incubated for 2 h with 0.5 mCi/ml of [³ H]thymidine.After aspiration and washing, the cells were lysed in 0.03% SDS. Thelysates were then treated with 10% trichloroacetic acid, and the [³H]thymidine incorporation measured.

Results

Overexpression of PC-1 in transfected cultured cells reduces insulinstimulated tyrosine kinase activity. MCF-7 cells, a human breastcarcinoma cell line that has been utilized for investigating insulinaction, were transfected with an expression plasmid containing PC-1cDNA. Control cells had a PC-1 activity of 10±5 nmoles/mg/min whichincreased to 405±35 nmoles/mg/min in transfected cells. Overexpressionof this protein did not alter insulin receptor binding (FIG. 6A). WithPC-1 overexpression, there was marked inhibition of insulin receptortyrosine kinase activity (FIG. 6B), as measured by both insulin receptorβ-subunit autophos-phorylation, and phosphorylation of the intracellularprotein, insulin receptor substrate-1 (IRS-I), Myers et al., Diabetes,42:643-650 (1993). In controls, effects of insulin on both functionswere observed at 1 nM, and maximal effects were observed at 10-100 nM.In transfected cells, effects were detected at 10 nM insulin, and at 100nM the effect of insulin was one half that of controls. In MCF-7 cells,insulin stimulates [³ H]thymidine incorporation into DNA, Myers et al.,supra) (FIG. 6c). In control MCF-7 cells, this function was halfmaximally stimulated at approximately 8 fold lower insulinconcentrations than in cells overexpressing PC-1 .

Example 8 Development of Anti-PC-1 Monoclonal Antibody Agents

A group of three Balb/c female mice (Charles River BreedingLaboratories, Wilmington, Mass.) were injected with 5 μg/dose ofpurified PC-1 in 100 μl Detox adjuvant (RIBI ImmunoChem Res. Inc.,Hamilton, Mont.) by intraperitoneal injection on days 0, 3, 7, 10, and14. On day 17 the animals were sacrificed, their spleens were removedand the lymphocytes fused with the mouse myeloma line 653, Kearney etal., J. Immunol., 123:1548 (1979) using 50% polyethylene glycol 4000 byan established procedure (Oi and Herzenberg, in Selected Methods inCellular Immunology, B. Mishel and S. Schiigi, eds., p. 351, W. J.Freeman Co., San Francisco, Calif., (1980). The fused cells were platedinto 96-well microtiter plates at a density of 2×10⁵ cells/well followedby HAT selection, Littlefield, J. W., Science, 145:709 (1964)) on day 1post fusion.

Immobilized hybridoma culture supernatants were reacted withbiotinylated PC-1. The wells positive for anti-PC-1 antibodies wereexpanded for further study. These cultures remained stable whenexpanded. Cell lines were cloned by limiting dilution and cryopreserved.The parental cultures were isotyped and assayed for their ability tocapture PC-1 and to neutralize in vitro PC-1 activity.

Initially, 13 hybridomas were identified producing antibodies that couldbind human PC-1. Of those, one hybridoma designated 4H9.3C7.1C5 wasfurther characterized.

The specificity of the antibody produced by the 4H9.3C7.1C5 hybridomawas tested by direct incubation with PC-1 in a standard ELISA. In thisassay wells of a flat bottomed 96 well plate were coated overnight at 4°C. with 100 μl of a solution containing purified human PC-1 atapproximately 1 μg/ml in carbonate buffer, pH 9.6. The plate has blockedfor 1 h with a solution of 1% BSA in PBS, then washed with wash buffer(0.05% Tween-20 in PBS). Monoclonal antibody purified from ascitesgenerated from hybridoma 4H9.3C7.1C5 as well as isotype matched controlswere added to the wells and the plate was incubated for 1-2 hours atroom temperature. The wells were washed and a solution containingapproximately 1 μg/ml horseradish peroxidase (HRP)-conjugated goatanti-mouse specific antibodies (Cappel, Westchester, Pa.), 0.5% BSA and0.25% NP-40, in wash buffer was added and the plates incubated for 2 h.After washing, the plates were developed by addition of a solutioncontaining 0.4 μg.ml o-phenylenediamine dihydrochloride plus 0.4/μl/ml30% hydrogen peroxide. The reaction was stopped by the addition of 2Nsulfuric acid. The color reaction was measured at 490 nm with anautomated ELISA plate reader.

The results of this assay showed that monoclonal antibody from hybridoma4H9.3C7.1C5 bound specifically to PC-1, and not appreciably to otherproteins.

The isotype of the antibody produced by 4H9.3C7.1C5 was determined byELISA. Hybridoma cell culture supernatants were added to the wells ofmicrotiter plates that had previously been coated with PC-1. Thecaptured anti-PC1 antibody was incubated with different isotype specificbiotin conjugated goat anti-mouse immunoglobulins (Zymed, South SanFransicso, Calif.), and the binding of the conjugated antibodies to theanti PC-1 monoclonal antibody was determined by the addition ofstreptaviden conjugated with HRP. The color reaction was measured at 405nm with an ELISA plate reader after addition of substrate.

By this method the isotype of the antibody was determined to be IgG1.

Western blot analysis was conducted according to the followingprocedure:

a) PC-1 obtained from transfected cells was subjected toSDS-polyacrylamide gel electrophoresis under reducing and non-reducingconditions with the appropriate controls and molecular weight standards.

b) the protein bands were transferred to solid support membranes(nitrocellulose) via semi-dry electrophoretic transfer (IntegratedSeparation Systems).

c) the membranes containing the transferred proteins were blocked with1% non-fat dry milk proteins for 2 hr or other blocking material such asbovine serum albumin and gelatin.

d) the membranes were then reacted with anti-PC-1 antibody and isotypematched control antibodies for 2 hr.

e) the membranes were washed with phosphate buffered saline containing0.05% Tween-20.

f)the membranes were then reacted with HRP-labelled anti-murineantibodies.

g) the membranes were washed.

h)the membranes were incubated with a chemi-luminescent substrate (ECLKit, Amersham) for 1 minute, exposed to x-ray film for 20-120 secondsand the film developed.

In this Western blot analysis, monoclonal antibodies obtained fromhybridoma 4H9.3C7.1C5 detected a non-reduced band at approximately 260kD corresponding to the expected molecular weight of the non-reducedPC-1. The antibody did not detect a band corresponding to the reducedform of the PC-1, suggesting that the monoclonal antibody recognizes aconformational epitope of PC-1.

The antibody was also able to recognize PC-1 expressing cells by flowcytometry. In this procedure MCF 7 cells transfected to express PC-1 ontheir surface were incubated with anti-PC-1 antibody for 20 min. at 4°C. The cells were washed free of excess anti-PC-1 and incubated withgoat anti mouse antibody labelled with fluorescein isothiocyanate (FITC)(TAGO) . The cells were incubated for 20 min. on ice and washed free ofexcess FITC labelled antibody. The cells were fixed in 0.05%paraformaldehyde. Labelled cells were detected using a FACSCAN (BectonDickenson).

The antibodies are appropriately used in an assay to detect PC-1expression or PC-1 over expression. The assay can be conducted on anysample suspected of containing human PC-1. Such a sample, for instance,contains dermal fibroblasts of a patient exhibiting insulin resistance.The cells are analyzed by flow cytometry and compared to cells of anormal individual. Alternatively, the cells are lysed with anappropriate lysing reagent and mixed with a known amount of aradio-labelled or otherwise tagged PC-1. In this assay, anti-PC-1antibodies are mixed with the sample containing the tagged PC-1 and theantibody precipitated using, for instance, a protein A reagent. TaggedPC-1 is measured and a reduction in signal is an indication of theamount of PC-1 present in the sample. This is compared to standardsample for an indication of the amount of PC-1 expressed in the sample.

Example 9 Determination of Affinities of Monoclonal Antibody Agents

The solid-phase radioimmunoassay procedure described by Mariani et al.,J. Immunol. Methods, 71: 43 (1984) is used to determine the affinitiesof the inhibitor specific monoclonal antibodies. Briefly, purifiedanti-inhibitor monoclonal antibodies are coated on Immunlon 2"Removawell" strips in pH 9.6 carbonate buffer for 18 hours at 4° C. Thewells are washed and blocked as described above. 40,000 CPM/well ofeither ¹²⁵ I-inhibitor (R & D Systems), in 50 μl PBSG, is added to2-fold serial dilutions of non-labeled inhibitor ranging from 2500 to9.7 ng/well, in 50 μl PBSG. The resulting mixture is incubated for 18hours at 4° C. The wells are washed and counted as described above andthe affinity constants determined by Scatchard analysis (Munson andPollard, supra), which yields similar results as the non-linearregression analysis of Antoni and Mariani, supra.

Example 10 Animal Models

In muscle and fat from male Wistar fatty rats, an animal model ofinsulin resistance and NIDDM, Greene et al., Obesity Res. 2:432-443(1994), insulin receptor content and insulin receptor tyrosine kinaseactivity is decreased (Greene et al., supra). In these two tissues, whencompared to controls, PC-1 content is elevated by 54 and 74%respectively. Antibodies described herein are injected into these ratsand it is expected that their PC-1 activity, as it affects insulinreceptor tyrosine kinase activity will be decreased.

Deposit of Materials

The following cultures are being deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC):

    ______________________________________                                        Hybridoma     ATCC No.  Deposit Date                                          ______________________________________                                        4H9.3C7       HB11957   July 19, 1995                                         ______________________________________                                    

This deposit is being made under the provisions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures for 30 years fromthe date of deposit. The organisms will be made available by ATCC underthe terms of the Budapest Treaty, and subject to an agreement betweenGenentech, Inc. and ATCC, which assures permanent and unrestrictedavailability of the progeny of the cultures to the public upon issuanceof the pertinent U.S. patent.

The assignee of the present application agrees that if the cultures ondeposit should die or be lost or destroyed when cultivated undersuitable conditions, they will be promptly replaced on notification witha viable specimen of the same culture. Availability of the depositedstrains are not to be construed as a license to practice the inventionin contravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the cultures deposited, sincethe deposited embodiments are intended as an illustration of an aspectof the invention and any cultures that are functionally equivalent arewithin the scope of this invention. The deposit of material herein doesnot constitute an admission that the written description hereincontained is inadequate to enable the practice of any aspect of theinvention, including the best mode thereof, nor is it to be construed aslimiting the scope of the claims to the specific illustration that itrepresents.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 1                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 925 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  Met Glu Arg Asp Gly Cys Ala Gly Gly Gly - #Ser Arg Gly Gly Glu            1               - #5                  - #10                  - #15          - -  Gly Gly Arg Ala Pro Arg Glu Gly Pro Ala - #Gly Asn Gly Arg Asp                            - #20                  - #25                  - #30          - -  Arg Gly Arg Ser His Ala Ala Glu Ala Pro - #Gly Asp Pro Gln Ala                            - #35                  - #40                  - #45          - -  Ala Ala Ser Leu Leu Ala Pro Met Asp Val - #Gly Glu Glu Pro Leu                            - #50                  - #55                  - #60          - -  Glu Lys Ala Ala Arg Ala Arg Thr Ala Lys - #Asp Pro Asn Thr Tyr                            - #65                  - #70                  - #75          - -  Lys Val Leu Ser Leu Val Leu Ser Val Cys - #Val Leu Thr Thr Ile                            - #80                  - #85                  - #90          - -  Leu Gly Cys Ile Phe Gly Leu Lys Pro Ser - #Cys Ala Lys Glu Val                            - #95                 1 - #00                 1 - #05        - -  Lys Ser Cys Lys Gly Arg Cys Phe Glu Arg - #Thr Phe Gly Asn Cys                           110 - #                115 - #                120             - -  Arg Cys Asp Ala Ala Cys Val Glu Leu Gly - #Asn Cys Cys Leu Asp                           125 - #                130 - #                135             - -  Tyr Gln Glu Thr Cys Ile Glu Pro Glu His - #Ile Trp Thr Cys Asn                           140 - #                145 - #                150             - -  Lys Phe Arg Cys Gly Glu Lys Arg Leu Thr - #Arg Ser Leu Cys Ala                           155 - #                160 - #                165             - -  Cys Ser Asp Asp Cys Lys Asp Lys Gly Asp - #Cys Cys Ile Asn Tyr                           170 - #                175 - #                180             - -  Ser Ser Val Cys Gln Gly Glu Lys Ser Trp - #Val Glu Glu Pro Cys                           185 - #                190 - #                195             - -  Glu Ser Ile Asn Glu Pro Gln Cys Pro Ala - #Gly Phe Glu Thr Pro                           200 - #                205 - #                210             - -  Pro Thr Leu Leu Phe Ser Leu Asp Gly Phe - #Arg Ala Glu Tyr Leu                           215 - #                220 - #                225             - -  His Thr Trp Gly Gly Leu Leu Pro Val Ile - #Ser Lys Leu Lys Lys                           230 - #                235 - #                240             - -  Cys Gly Thr Tyr Thr Lys Asn Met Arg Pro - #Val Tyr Pro Thr Lys                           245 - #                250 - #                255             - -  Thr Phe Pro Asn His Tyr Ser Ile Val Thr - #Gly Leu Tyr Pro Glu                           260 - #                265 - #                270             - -  Ser His Gly Ile Ile Asp Asn Lys Met Tyr - #Asp Pro Lys Met Asn                           275 - #                280 - #                285             - -  Ala Ser Phe Ser Leu Lys Ser Lys Glu Lys - #Phe Asn Pro Glu Trp                           290 - #                295 - #                300             - -  Tyr Lys Gly Glu Pro Ile Trp Val Thr Ala - #Lys Tyr Gln Gly Leu                           305 - #                310 - #                315             - -  Lys Ser Gly Thr Phe Phe Trp Pro Gly Ser - #Asp Val Glu Ile Asn                           320 - #                325 - #                330             - -  Gly Ile Phe Pro Asp Ile Tyr Lys Met Tyr - #Asn Gly Ser Val Pro                           335 - #                340 - #                345             - -  Phe Glu Glu Arg Ile Leu Ala Val Leu Gln - #Trp Leu Gln Leu Pro                           350 - #                355 - #                360             - -  Lys Asp Glu Arg Pro His Phe Tyr Thr Leu - #Tyr Leu Glu Glu Pro                           365 - #                370 - #                375             - -  Asp Ser Ser Gly His Ser Tyr Gly Pro Val - #Ser Ser Glu Val Ile                           380 - #                385 - #                390             - -  Lys Ala Leu Gln Arg Val Asp Gly Met Val - #Gly Met Leu Met Asp                           395 - #                400 - #                405             - -  Gly Leu Lys Glu Leu Asn Leu His Arg Cys - #Leu Asn Leu Ile Leu                           410 - #                415 - #                420             - -  Ile Ser Asp His Gly Met Glu Gln Gly Ser - #Cys Lys Lys Tyr Ile                           425 - #                430 - #                435             - -  Tyr Leu Asn Lys Tyr Leu Gly Asp Val Lys - #Asn Ile Lys Val Ile                           440 - #                445 - #                450             - -  Tyr Gly Pro Ala Ala Arg Leu Arg Pro Ser - #Asp Val Pro Asp Lys                           455 - #                460 - #                465             - -  Tyr Tyr Ser Phe Asn Tyr Glu Gly Ile Ala - #Arg Asn Leu Ser Cys                           470 - #                475 - #                480             - -  Arg Glu Pro Asn Gln His Phe Lys Pro Tyr - #Leu Lys His Phe Leu                           485 - #                490 - #                495             - -  Pro Lys Arg Leu His Phe Ala Lys Ser Asp - #Arg Ile Glu Pro Leu                           500 - #                505 - #                510             - -  Thr Phe Tyr Leu Asp Pro Gln Trp Gln Leu - #Ala Leu Asn Pro Ser                           515 - #                520 - #                525             - -  Glu Arg Lys Tyr Cys Gly Ser Gly Phe His - #Gly Ser Asp Asn Val                           530 - #                535 - #                540             - -  Phe Ser Asn Met Gln Ala Leu Phe Val Gly - #Tyr Gly Pro Gly Phe                           545 - #                550 - #                555             - -  Lys His Gly Ile Glu Ala Asp Thr Phe Glu - #Asn Ile Glu Val Tyr                           560 - #                565 - #                570             - -  Asn Leu Met Cys Asp Leu Leu Asn Leu Thr - #Pro Ala Pro Asn Asn                           575 - #                580 - #                585             - -  Gly Thr His Gly Ser Leu Asn His Leu Leu - #Lys Asn Pro Val Tyr                           590 - #                595 - #                600             - -  Thr Pro Lys His Pro Lys Glu Val His Pro - #Leu Val Gln Cys Pro                           605 - #                610 - #                615             - -  Phe Thr Arg Asn Pro Arg Asp Asn Leu Gly - #Cys Ser Cys Asn Pro                           620 - #                625 - #                630             - -  Ser Ile Leu Pro Ile Glu Asp Phe Gln Thr - #Gln Phe Asn Leu Thr                           635 - #                640 - #                645             - -  Val Ala Glu Glu Lys Ile Ile Lys His Glu - #Thr Leu Pro Tyr Gly                           650 - #                655 - #                660             - -  Arg Pro Arg Val Leu Gln Lys Glu Asn Thr - #Ile Cys Leu Leu Ser                           665 - #                670 - #                675             - -  Gln His Gln Phe Met Ser Gly Tyr Ser Gln - #Asp Ile Leu Met Pro                           680 - #                685 - #                690             - -  Leu Trp Thr Ser Tyr Thr Val Asp Arg Asn - #Asp Ser Phe Ser Thr                           695 - #                700 - #                705             - -  Glu Asp Phe Ser Asn Cys Leu Tyr Gln Asp - #Phe Arg Ile Pro Leu                           710 - #                715 - #                720             - -  Ser Pro Val His Lys Cys Ser Phe Tyr Lys - #Asn Asn Thr Lys Val                           725 - #                730 - #                735             - -  Ser Tyr Gly Phe Leu Ser Pro Pro Gln Leu - #Asn Lys Asn Ser Ser                           740 - #                745 - #                750             - -  Gly Ile Tyr Ser Glu Ala Leu Leu Thr Thr - #Asn Ile Val Pro Met                           755 - #                760 - #                765             - -  Tyr Gln Ser Phe Gln Val Ile Trp Arg Tyr - #Phe His Asp Thr Leu                           770 - #                775 - #                780             - -  Leu Arg Lys Tyr Ala Glu Glu Arg Asn Gly - #Val Asn Val Val Ser                           785 - #                790 - #                795             - -  Gly Pro Val Phe Asp Phe Asp Tyr Asp Gly - #Arg Cys Asp Ser Leu                           800 - #                805 - #                810             - -  Glu Asn Leu Arg Gln Lys Arg Arg Val Ile - #Arg Asn Gln Glu Ile                           815 - #                820 - #                825             - -  Leu Ile Pro Thr His Phe Phe Ile Val Leu - #Thr Ser Cys Lys Asp                           830 - #                835 - #                840             - -  Thr Ser Gln Thr Pro Leu His Cys Glu Asn - #Leu Asp Thr Leu Ala                           845 - #                850 - #                855             - -  Phe Ile Leu Pro His Arg Thr Asp Asn Ser - #Glu Ser Cys Val His                           860 - #                865 - #                870             - -  Gly Lys His Asp Ser Ser Trp Val Glu Glu - #Leu Leu Met Leu His                           875 - #                880 - #                885             - -  Arg Ala Arg Ile Thr Asp Val Glu His Ile - #Thr Gly Leu Ser Phe                           890 - #                895 - #                900             - -  Tyr Gln Gln Arg Lys Glu Pro Val Ser Asp - #Ile Leu Lys Leu Lys                           905 - #                910 - #                915             - -  Thr His Leu Pro Thr Phe Ser Gln Glu Asp                                                  920 - #                925                                  __________________________________________________________________________

What is claimed is:
 1. A method for neutralizing the effect of PC-1comprising contacting insulin receptor tyrosine kinase inhibitor, classII human membrane glycoprotein PC-1 with an antibody specific for PC-1in an amount sufficient to effect neutralization.
 2. The methodaccording to claim 1 in which the antibody is a monoclonal antibody. 3.The method according to claim 1 in which the method is performed in amammal.
 4. A method for neutralizing the effect of PC-1 comprisingcontacting insulin receptor tyrosine kinase inhibitor, class II humanmembrane glycoprotein PC-1 in vitro with an antibody specific for PC-1in an amount sufficient to effect neutralization.
 5. The methodaccording to claim 4 in which the antibody is a monoclonal antibody.